Using small animal PET, we have shown that it is possible to detect a statistically significant decrease in [11
in the rat striatum following I.P. or I.V. alcohol treatment. We infer this decrease in BPND
to reflect increase in striatal DA. The largest and most significant increase in DA concentration (
) was seen in Wistars (n = 14) given 3.0 g/kg I.P. alcohol. It should also be noted that while significant decreases in BPND
were found with 1.0 g/kg I.V. alcohol, the cohort was small (n = 3) and thus the results are highly preliminary but encouraging. We found no statistical effect of I.P. alcohol dose on
. It is likely that larger cohorts would be necessary to examine dose dependent effects of alcohol on BPND
Complications in collecting blood from the tail vein prevented us from measuring BAC in all animals. To circumvent the problems related to tail vein sampling, we collected blood samples from the lateral saphenous vein in some animals. Regardless of which vein was used, it was difficult to collect blood. Under anesthesia and alcohol administration blood pressure appeared to decrease and blood viscosity appeared to increase (based on visual observation). From the blood samples available, we found a positive correlation between
and BAC. There was also high variability in BAC measured at the 2.25g/kg alcohol dose. This variability may reflect errors in the actual alcohol content of the injected solution or errors in the I.P. injection. Though steps were taken to insure that both of these procedures were carried-out correctly, we cannot rule them out as sources of variability. Six out of the 7 animals with BAC>200mg% showed
greater than 8% (i.e. increase in DA concentration). Notwithstanding the high degree of variability in BAC produced by a given alcohol dose, this data may suggest that BAC needs to be greater than 200mg% to reliably produce a detectable increase in DA concentration under the present imaging protocol.
As stated above, alcohol-preferring (P) rats are an established animal model of alcoholism. P rats’ increased predilection to consume alcohol is thought to be due, in part, to lower basal DA concentrations (Murphy et al., 1982
) and to increased DA release in the striatum in response to alcohol (Weiss et al., 1993
). Free-choice drinking studies have shown that alcohol induces significantly greater DA release in P rats than in Wistars (Weiss et al., 1993
). Similarly, I.P. administration of alcohol results in greater striatal DA release (measured by microdialysis) in an alcohol preferring rat line compared with an alcohol abstaining line (University of Chile bibulous vs. Abstainer rats) (Bustamante et al., 2008
). Thus, we expected that P rats would exhibit a greater DA response to alcohol (greater
) than Wistars. This was not observed (). However, given the limited size of the P-IP-3G group (n = 4), it was not possible to make a definitive comparison between them and the comparably treated Wistars (WIS-IP-3G).
We did find that P rats had lower resting [11
C]raclopride binding potential (
) than Wistars. Autoradiographic studies have shown that P rats have lower striatal D2 receptor densities than alcohol non-preferring (NP) rats (McBride et al., 1993
), and recent imaging studies have shown the P rats have lower resting [11
C]raclopride binding potential than NP rats (Thanos et al., 2004
). If we assume that the non-displaceable tissue free fraction (fP
) and disassociation constant (KD
) of [11
C]raclopride for D2
receptors are the same across lines, we can interpret [11
, (Innis et al., 2007
)) as a measure of D2 receptor density (Bavail
). Because Ps and NPs are based on Wistars, one might speculate that through selective breeding P rats have acquired lower D2 receptor density than Wistars. Thus, our findings are consistent with both the work of Thanos et al. and McBride et al.
In the present study, almost all P rats were given lower doses of alcohol than Wistars because, in our hands, P rats were prone to fatal overdose by the combination of anesthesia and high dose alcohol. Anesthesia induction in P rats was variable. Unlike Wistars, for whom full anesthesia was consistently induced with 5% isoflurane, some P rats would fluctuate between consciousness and unconsciousness repeatedly prior to imaging. Additionally, it was considerably more difficult to locate the tail vein for tracer injections in P rats than in Wistar rats. This may be due to lower resting blood pressure in P rats, as has been reported in another line of alcohol-preferring rats (AA) compared to their alcohol-avoiding counterparts (ANA) (Linkola et al., 1980
). Consistent with the literature, P rats were apparently less sensitive to the central depressant effects of alcohol relative to Wistars (Kurtz et al., 1996
; Lumeng et al., 1982
). P rats tended to regain motor control and normal behavior sooner than Wistars after administration of comparable doses of alcohol and anesthesia.
Isoflurane was chosen for these studies because it is easy to provide and, as a gas, easily used to maintain anesthesia throughout the scan. No difference has been shown in [11
C]raclopride binding between awake and isoflurane anesthetized monkeys (Tsukada et al., 2002
), but several studies have shown that isoflurane effects the DA system. In rats, isoflurane and halothane (at similar doses to what was used in here) have been found to decrease extracellular DA and increase DA metabolites (Adachi et al., 2000
; Adachi et al., 2005
). In our studies, this would result in an attenuation of the effect of alcohol on BPND
. A more concerning confound would be if isoflurane enhanced the decrease in BPND
due to alcohol, as has been seen in monkeys with nicotine and methamphetamine (Tsukada et al., 2002
). Given this, we cannot discount that isoflurane may have had an effect on the DA system of the animals we were studying.
No attenuation correction was applied to the scans in this study. Given that all scans for each animal were completed within a short time frame (3–4 weeks, reducing animal growth as a confounding factor), we do not believe that lack of attenuation correction negatively affected our analysis. Furthermore, our semi-stereotactic holder (made of acrylic plastic, which has a low linear attenuation coefficient) provided highly reproducible repositioning, making attenuation effects negligible (Cheng et al., 2009
Injection of an equal volume of saline was used in the current study to control for the stress associated with I.P. injection. Although the mesolimbic DA system has been shown to be involved in perception of pain and response to noxious stimuli in both awake and anesthetized animals (Gear et al., 1999
; Schultz and Romo, 1987
; Ungless et al., 2004
), data from the saline scans indicated that there was no significant effect of I.P. injection of inert fluid on BPND
. This supports our conclusions that the observed decreases in BPND
(increased DA) were primarily due to the pharmacological effects of alcohol.
Seven Wistars were implanted with jugular catheters for I.V. infusion of alcohol. Problems maintaining catheter patency reduced the usefulness of this approach; only 3 of the 7 animals implanted were patent for I.V. infusions (WIS-IV-1G). Nevertheless, this study design provided an opportunity to assess the effect of surgical cannula implantation on striatal DA concentration. Wistars with implanted ports had significantly higher
than those without ports. Although animals with ports did not exhibit signs of pain during the course of the study (e.g. self-mutilation, loss of appetite), our findings may reflect the effect of increased stress on D2 receptor density or basal DA level. Rats with ports were housed singly after surgery and during the PET scan experiments (~3 weeks for the duration of experiments), which may have introduced isolation stress. It is possible that chronic, low-level stress increases
(i.e. D2 receptor availability), either through a reduction in basal DA concentration (Gambarana et al., 1999
) or through up-regulation of D2 receptor density (Lucas et al., 2007
). The suggestion that standard housing and surgery could affect BPND
is relevant to future experiment design and warrants further investigation.
In summary, we were able to detect alcohol-induced changes in [11
in the striatum of anesthetized Wistar and P rats using small animal PET and feel confident that these observed changes reflect the effects of alcohol on DA release in the rat striatum. Our results suggest that achieving threshold BAC may be necessary to provoke DA release that is measurable with PET. We also detected differences in basal D2 receptor availability between Wistar and P rats and observed what may be an effect of chronic stress on D2 receptor availability in Wistars. Further work is needed with larger cohorts to confirm the ability to image DA release in P rats. A full comparison of modes of alcohol delivery (I.V. vs. I.P.) and their effect on PET imaging of alcohol-induced DA release would be useful. Additional work must also address what ranges of alcohol dose are physiologically relevant and likely to produce adequate BAC to realize a consistently detectable